The availability of detailed structures of biomolecules can expedite the translation of genomic and proteomic information into therapeutic and prophylactic interventions. Unfortunately, the size, pleiomorphism and/or conformational flexibility of some proteins renders them refractory to conventional approaches to obtaining structure, such as x-ray crystallography or NMR spectroscopy. One such example is the trimeric human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein complex, which mediates the entry of the virus into the host cell. The unliganded HIV-1 Env glycoprotein complex exists in a high-energy state; upon binding to the CD4 and CCR5 receptors, the HIV-1 Env glycoproteins assume lower-energy conformations. These conformational transitions in the HIV-1 Env glycoprotein complex ultimately result in the fusion of the viral and target cell membranes. As the only virus-specific protein exposed on the HIV-1 membrane, the Env glycoprotein trimer represents a major target for entry inhibitors, including small molecules, peptides and neutralizing antibodies. Unfortunately, conformational flexibility, the lability of the unliganded state, and a high degree of glycosylation have slowed structural studies of the Env glycoprotein trimer. This gap in knowledge represents a major barrier to progress in HIV-1 entry inhibition and vaccine immunogen design. The proposed studies will utilize single-particle cryoelectron microscopy to yield structures of membrane-anchored HIV-1 Env trimers in different conformations at near-atomic (3.5 - 5 E) resolution. The Specific Aims of this application are: 1) To prepare membrane-anchored HIV-1 Env glycoprotein trimers that are suitable for high-resolution structure determination by single-particle cryoelectron microscopy and to solve the structure of the unliganded HIV-1 Env trimer; 2) To investigate the structure of the HIV-1 gp41 cytoplasmic tail and its potential contribution to the structure of the Env ectodomain; and 3) To solve the structure of the CD4-bound conformation of the HIV-1 Env glycoprotein trimer. These studies will yield detailed snapshots of two key stages in the process of HIV-1 entry, providing a framework for understanding the dynamic aspects of the Env glycoprotein membrane-fusing machine. Conserved, functionally important structures on the HIV-1 Env glycoprotein complex that can serve as targets for drugs or vaccine-induced antibodies will be revealed. This information will transform our understanding of HIV-1 entry and should inspire new approaches to intervention. PUBLIC HEALTH RELEVANCE: The human immunodeficiency virus (HIV-1) envelope glycoprotein spike mediates the entry of the virus into the host cell, and therefore is an attractive target for inhibitors and vaccines. However, lack of information about the structure of the HIV-1 envelope glycoprotein spike has been a major barrier to progress in this area. We will develop methods in electron microscopy that allow a detailed structural blueprint of the HIV-1 envelope glycoprotein spike to be realized.